CN101624420A

CN101624420A - Group of novel cyclized antibiotic peptides containing gamma-core motif and preparation method as well as application thereof

Info

Publication number: CN101624420A
Application number: CN200910023033A
Authority: CN
Inventors: 罗晓星; 侯征; 吕军; 孟静茹; 白卉; 周颖; 薛小燕
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2009-06-24
Filing date: 2009-06-24
Publication date: 2010-01-13
Anticipated expiration: 2029-06-24
Also published as: CN101624420B

Abstract

The invention provides a group of novel cyclized antibiotic peptides containing gamma-core motif. The antibiotic peptides contain genetic evolution symbols and have the advantages of good antibiotic effects, low toxicity, good stability and good tolerance. The invention also discloses a solid-state chemical preparation and air oxidation method of the antibiotic peptides. The antibiotic peptides synthesized by the invention can be used for preparing the medicines against multi-resistance bacteria infection.

Description

One group of novel cyclized antibiotic that contains the gamma-core block and its production and application

Technical field

The present invention relates to the application of one group of novel cyclized antibiotic that contains the gamma-core block and preparation method thereof and the infection of treatment multi-drug resistant bacteria.

Background technology

So far, more than 600 antibacterial peptide (is referred to as positively charged ion host defense peptide again, AMPs) be proved pathogenic microorganisms such as to kill gram-positive microorganism, Gram-negative bacteria, virus, protozoon and fungi, and played a significant role at recruitment scavenger cell and enhancing innate immune system.Compare with traditional microbiotic, antibacterial peptide kill bacteria fireballing relate to a plurality of target spots in the bacterial cell more.They can be used alone as antiseptic-germicide, also can bring into play synergistic effect in conjunction with other microbiotic, perhaps as immunomodulator and in and the intracellular toxin material.It is worth noting most that it is chemical sproof fully that antibacterial peptide is acknowledged as impossible generation.Because it mainly be by the physics osmosis in bacterial membrane, microorganism be difficult to change after birth (phospholipid bilayer) structure of self, thereby makes microorganism be difficult for it is produced resistance.In addition, antibacterial peptide has a plurality of action target spots in microbe, reduces any one action target spot in the born of the same parents, and it is a lot of that its resistance is increased.Simultaneously, crossing drug resistant can not take place in antibacterial peptide and other antibacterial peptide or traditional microbiotic combined utilization.Antibacterial peptide is difficult for producing chemical sproof superior character, provides new field for people seek the ideal antibacterials, has brought new opportunity for the mankind defeat drug-resistant microorganism, and therefore antibacterial peptide also has the potentiality of huge research and development and application.

Although the natural antibacterial peptide that nature is found and artificial reconstructed, design synthetic antibacterial peptide is of a great variety, it is but very low that antibacterial peptide is applied to clinical success ratio.Its major cause is that antibacterial peptide also exists some problem demanding prompt solutions in research and development: the first, and toxicity problem is its major cause that can not be applied to whole body.Antibacterial peptide is mainly brought into play germicidal action by destroying bacterial cell membrane, and selectivity is not high, and nonselective cytotoxicity easily makes the erythrocyte membrane haemolysis that breaks.Too big as polymyxins and Gramicidin S in clinical use toxic, can not use as antiseptic-germicide or antiendotoxin agent whole body, can only be united use by the part.The second, stability problem is the second largest difficult point of its clinical application.Antibacterial peptide is the protein of small molecular weight, and easily by protease hydrolysis, the transformation period is short in the body.The 3rd, antibacterial peptide inside and outside activity there are differences.Although many antibacterial peptides demonstrate tangible anti-microbial activity external, under physiological salt concentration and blood plasma condition, the loss of activity of many antibacterial peptides.And some host defense peptide but can be brought into play anti-microbial effect by immunomodulatory in vivo in external no antibiotic activity.The 4th, the production cost costliness is to limit the immediate cause of its clinical application.The source of antibacterial peptide is very difficult: natural extract resource-constrained, separation purifying technique complexity, cost height; Chemosynthesis is with respect to microbiotic, and cost is also much higher, the industrialization difficulty; Gene engineering expression then anti-microbial activity poor, easily cause host cell to be committed suiside, be difficult to do expression system, and it is not high to express output with bacterium, virus commonly used.Therefore, being badly in need of transforming the native peptides that molecular weight is big, production cost is high is the artificial peptide that molecular weight is little, production cost is low relatively, thereby finally reduces its production cost.

Recently in containing many polypeptide such as the stable antimicrobial polypeptide of halfcystine, toxin, venom, kinocidins and host defense peptide, found a structure block---gamma-core block.It is a conservative three-dimensional structure block with genetic evolution sign, the bidirectional oriented structure of being made up of the specific amino acids sequence.Thanatin is the simplest antibacterial peptide of finding at present with gamma-core block.Gram-negative bacteria is had stronger anti-microbial activity, and toxicity is low, good stability.The present invention transforms the thanatin sequence by brachymemma, replacement, displacement and cyclisation, obtains having different antimicrobial spectrums, low toxicity, the stable novel cyclized antibiotic that contains the gamma-core block.

Summary of the invention

One of technical issues that need to address of the present invention provide one group of novel antibacterial peptide.

Two of the technical issues that need to address of the present invention provide the preparation method of one group of novel antibacterial peptide.

Three of the technical issues that need to address of the present invention are the application that disclose described antibacterial peptide.

Design of the present invention is as follows: the main thought of improvement and design is the genetic evolution sign (gamma-core block) that keeps thanatin, corresponding site sequence with 6 naturally occurring aminoacid sequences that contain gamma-core block antibacterial peptide β ridge are replaced thanatin obtains a series of new sequences; Cut out 8 amino acid of these sequences N-end, a series of new sequences of getting back; Cut out 1 amino acid of these sequence C-ends on this basis again, getting back only keeps a series of new sequence of gamma-core, and the C-end of above-mentioned all polypeptide chains all carries out amidation, and the equal cyclisation of two halfcystines of peptide intrachain forms a Gelucystine.Have different anti-microbial activities through screening following 37 sequences after transforming, and hemolytic toxicity not have variation substantially, be hopeful very much to be used for the treatment of the exploitation of the new drug of infectation of bacteria.

Antibacterial peptide provided by the invention is to design synthetic on to the sequence of natural antibacterial peptide, basis of structural analysis, and a series of sequences of they this are named as DS, and sequence is as follows:

Thanatin

DS01

DS02

DS03

DS04

DS05

DS06

DS07

DS08

DS09

DS10

DS11

DS12

DS13

DS14

DS15

DS16

DS17

DS18

DS19

DS20

DS21

DS22

DS23

DS24

DS25

DS26

DS27

DS28

DS29

DS30

DS31

DS32

DS33

DS34

DS35

DS36

DS37

The described novel cyclized antibiotic that contains the gamma-core block is 12,13 and 21 amino acid, and the whole amidations of C-terminal are modified, and the equal cyclisation of two halfcystines of peptide intrachain forms a Gelucystine.

One group of antibacterial peptide provided by the invention, its preparation method are that solid state chemistry is synthetic.Measure the molecular weight of polypeptide crude product earlier with the MALDI-TOF mass spectrograph; With the RP-HPLC chromatographic instrument polypeptide crude product is carried out separation and purification again; Measure the molecular weight of the pure product of polypeptide at last with the ESI mass spectrograph, and the pure product of polypeptide are carried out purity check with the RP-HPLC chromatographic instrument.For the polypeptide chain of cyclisation, adopt the method for atmospheric oxidation to form intramolecular disulfide bond.In the polypeptide oxidising process, constantly analyze the variation of polypeptide oxidation chromatographic peak and reduction chromatographic peak area with reverse high performance liquid chromatography (RP-HPLC); Monitor the process of polypeptide oxidation and the polypeptide after the oxidation is carried out the RP-HPLC purifying; Measure the molecular weight of the pure product of oxidation front and back polypeptide at last with the ESI mass spectrograph.

Adopting test tube method to detect the killing curve of polypeptide, is contrast with microbiotic Ampicillin Trihydrate and ceftazime, carries out the detection of fungicidal activity.The result shows that its fungicidal activity of antibacterial peptide that the present invention prepares is better than described two kinds of antibiotic fungicidal activities.The killing curve result shows that antibacterial peptide has the obvious sterilization effect to Gram-positive resistant organism methicillin-resistant staphylococcus epidermidis (MRSE), and along with the prolongation of action time, bacterial count reduces gradually.

Adopting test tube method to detect the dose effect curve of different concns polypeptide, is contrast with the microbiotic Ampicillin Trihydrate, carries out the detection of fungicidal activity.The concentration effect relational result shows that the different concns antibacterial peptide has significant killing action to MRSE, and has concentration dependence effect.

Antibacterial peptide also might act on high organism and comprise human body cell in efficient sterilizing, because the mode of action of antibacterial peptide mainly is that perforation makes the death of cell generation seepage on cytolemma.So can make red corpuscle generation seepage as its virose standard whether antibacterial peptide, if antibiotic Toplink makes the oxyphorase generation seepage in the red corpuscle, just can be by detecting OD ₅₄₀Value is determined toxic size.Therefore the present invention has also detected the hemolytic activity of antibacterial peptide to human erythrocyte, and experiment shows that when 128 μ g/ml the hemolysis rate value of antibacterial peptide still is zero, confirms that the hemolytic toxicity of antibacterial peptide antibacterial peptide of the present invention is minimum, they? partly imitate hemolytic concentration (HC ₅₀) be worth far above positive control Melittin.

For the stability of clear and definite polypeptide in blood plasma, we have carried out the experiment of 50% plasma stability, after the result shows that antibacterial peptide and blood plasma are hatched 6h, its MIC value is constant, show that they are stable in blood plasma, and the contrast antibacterial peptide S4 (1-16) the MIC value obviously increase, illustrate it in blood plasma by severely degrade.

The resistance experimental result proves, after MRSE and 15 generations of drug interaction, used microbiotic erythromycin, ceftriaxone and Oxazacillin all produced serious resistance, and antibacterial peptide is not all produced resistance.

Description of drawings

Now in conjunction with the accompanying drawings and embodiments content of the present invention is described further.

Fig. 1 is the MALDI-TOF mass spectrum of antibacterial peptide US34 crude product;

Fig. 2 is the ESI-MS mass spectrum of the pure product of antibacterial peptide US34;

Fig. 3 is the RP-HPLC color atlas of the pure product of antibacterial peptide US34;

Fig. 4 is the RP-HPLC color atlas behind the pure product atmospheric oxidation of the antibacterial peptide US32 different time;

Fig. 5 is the ESI-MS mass spectrum and the RP-HPLC color atlas of pure product before and after the antibacterial peptide US32 cyclisation;

Fig. 6 is the ESI-MS mass spectrum of the pure product of antibacterial peptide DS34;

Fig. 7 is the RP-HPLC color atlas of the pure product of antibacterial peptide DS34;

Fig. 8 is the killing curve of antibacterial peptide DS34;

Fig. 9 is the statistical graph that various dose DS34 effect rear plate clone forms experimental result;

Figure 10 is the haemolysis curve of antibacterial peptide DS34;

Figure 11 is the resistance experiment of antibacterial peptide DS34;

Figure 12 is the 50% plasma stability experiment of antibacterial peptide DS34.

Embodiment

Below by specific embodiment, be example with DS34, be described in further detail the present invention.

The preparation and the separation and purification of embodiment 1 antibacterial peptide.In the present embodiment, prepare DS1 to DS37, prepare Melittin and K4-S4 (1-16) simultaneously in contrast by above-mentioned sequence.Present embodiment is condensing agent with HOBt/DIC, on the Rink resin, adopts Fmoc protection alpha amino acid, from the polypeptide chain of C-end (carboxyl terminal) to the synthetic C-terminal amideization of N-end (aminoterminal) manual solid-phase.The synthetic polypeptide is measured the molecular weight of polypeptide crude product earlier with the MALDI-TOF mass spectrograph after excessive concentrations TFA shears; With the RP-HPLC chromatographic instrument polypeptide crude product is carried out separation and purification again; Measure the molecular weight of the pure product of polypeptide at last with the ESI mass spectrograph, and the pure product of polypeptide are carried out purity check with the RP-HPLC chromatographic instrument.Adopt the method for atmospheric oxidation to form intramolecular disulfide bond.

The sequence of Melittin:

Gly-Ile-Gly-Ala-Val-Leu-lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH ₂。

The sequence of K4-S4 (1-16):

Ala-Leu-Trp-Lys-Thr-Leu-Leu-Lys-Lys-Val-Leu-Lys-Ala-Ala-Ala-Lys-NH ₂。

Concrete testing sequence is as follows.

The first step, the solid phase synthesis of antibacterial peptide (is example with No. 34 samples).The polypeptide of preparation holds the N end to carry out one by one from C.At first weighing 0.5gRink Amide-AM resin dress post adds 2.5ml methylene dichloride (CHCl ₂), make its swelling, 20% piperidines/dimethyl formamide (DMF) solution deprotection, DMF cleans.First amino acid, hydroxybenzotriazole (HOBt) and the DIC (DIC) of 9-tablet held before the breast by officials methoxycarbonyl (Fmoc) protection are dissolved in an amount of DMF, and the resin behind activation back and the deprotection is at post cocycle coupled reaction 30min～2h, and DMF washs.Repeat the process of above activation, condensation, deprotection and washing with latter linked amino acid, finish up to preparation.

Second step, the shearing of antibacterial peptide (is example with No. 34 samples).Take off reacted resin, add cutting liquid (90% trifluoroacetic acid, 5% thioanisole, 3% methyl-phenoxide, 2% dithioglycol), room temperature reaction 3-4 hour, filter, the precooling anhydrous diethyl ether that adds 10 times of volumes in the filtrate, 3500 rev/mins centrifugal 5 minutes, collecting precipitation and drying at room temperature.

In the 3rd step, the MALDI-TOF mass spectrum of antibacterial peptide is identified (is example with No. 34 samples).The ferric oxide dispersion liquid point of getting 0.5 μ l polypeptide sample solution on the MALDI target plate, the organic substrate solution of last again 0.5 μ L, treat the sampling liquid drying, crystallization on the target plate after, put target plate into mass spectrograph, carry out mass spectroscopy.The result as shown in Figure 1, the US34 of preparation analyzes through MALDI-TOF, the theoretical value (1641.9KD) that molecule measuring definite value (1642.0KD) and the peptide sequence that shows in mass spectrum calculates is consistent, illustrates that the polypeptide for preparing is the antimicrobial peptide of design.

The 4th step, the RP-HPLC purifying of antibacterial peptide (is example with No. 34 samples).The a certain amount of dried polypeptide of weighing is dissolved in 0.1% trifluoroacetic acid, and sample preparation is after reverse post gradient separations (elutriant is the acetonitrile that contains the 20%-80% of 0.1% trifluoroacetic acid) is collected elution peak.

In the 5th step, the ESI-MS mass spectrum of antibacterial peptide is identified (is example with No. 34 samples).The a certain amount of dried polypeptide of weighing is dissolved in methyl alcohol, detects with electron spray ionisation negative ion, multiple-reaction monitoring mode after the sample preparation.The result as shown in Figure 2, US34 behind the purifying identifies that through ESI-MS the theoretical value (1641.9KD) that molecule measuring definite value (1641.6KD) and the peptide sequence that shows calculates is consistent, illustrates that the polypeptide behind the purifying is US34 in mass spectrum.

In the 6th step, the RP-HPLC of antibacterial peptide analyzes (is example with No. 34 samples).The a certain amount of dried polypeptide of weighing is dissolved in 0.1% trifluoroacetic acid, and sample preparation is calculated the purity of the pure product of polypeptide after reverse chromatograms post gradient analysis (elutriant is the acetonitrile that contains the 20%-80% of 0.1% trifluoroacetic acid).The result as shown in Figure 3, US34 behind the purifying analyzes through RP-HPLC, shows that in color atlas its purity is 98.11%, obviously reaches more than 95%.

The 7th step, the intramolecular cyclization of antibacterial peptide (is example with No. 32 samples).With deionized water (pH 6.5-8.5) dissolving polypeptide, making peptide concentration is 0.01-0.1mg/ml; Continue blowing air in the solution of reaction; Detect the oxidation process of polypeptide chain with HPLC every 3h.Among Fig. 4, A, B, C are respectively the color atlas result before, during and after the sample oxidation No. 32, therefrom as can be seen, prolongation along with the atmospheric oxidation time, the oxidation peak area increases gradually in No. 32 samples, and the reduction peak area reduces gradually, illustrates that the content of cyclisation polypeptide increases gradually.When the most of oxidation of sample is intact (about 24h of reaction times), termination reaction.Shown in Figure 5, top figure is color atlas and the mass spectrum of US32 before the oxidation; Following figure is color atlas and the mass spectrum of DS32 after the oxidation.As can be seen from the figure, oxidation is forward and backward, and the purity of No. 32 purified samples is respectively 96.27% and 96.04%, all reaches more than 95%.The molecular weight of forward and backward US32 of oxidation and DS32 is respectively 1495.0KD and 1493.0KD, only differs 2, shows correct formation intramolecular disulfide bond.

The 8th step, the RP-HPLC purifying of polypeptide after the cyclisation (is example with No. 34 samples).The a certain amount of dried polypeptide of weighing is dissolved in 0.1% trifluoroacetic acid, and sample preparation is after reverse post gradient separations (elutriant is the acetonitrile that contains the 20%-80% of 0.1% trifluoroacetic acid) is collected elution peak.

In the 9th step, the ESI-MS mass spectrum of polypeptide is identified (is example with No. 34 samples) after the cyclisation.The a certain amount of dried polypeptide of weighing is dissolved in methyl alcohol, detects with electron spray ionisation negative ion, multiple-reaction monitoring mode after the sample preparation.The result as shown in Figure 6, DS34 behind the purifying identifies that through ESI-MS the theoretical value (1639.9KD) that molecule measuring definite value (1640.0KD) and the peptide sequence that shows calculates is consistent, illustrates that the polypeptide behind the purifying is DS34 in mass spectrum.

In the tenth step, the RP-HPLC of polypeptide analyzes (is example with No. 34 samples) after the cyclisation.The a certain amount of dried polypeptide of weighing is dissolved in 0.1% trifluoroacetic acid, and sample preparation is calculated the purity of the pure product of polypeptide after oppositely post gradient analysis (elutriant is the acetonitrile that contains the 20%-80% of 0.1% trifluoroacetic acid).The result as shown in Figure 7, DS34 behind the purifying analyzes through RP-HPLC, shows that in color atlas its purity is 97.28%, obviously reaches more than 95%.

The mensuration of embodiment 2 antibacterial peptide killing curves.

The first step is got 4 of aseptic Erlenmeyer flasks, is labeled as " growth control pipe, medicine 1, medicine 2 and medicine 3 (positive control drug) " respectively.Every bottle adds the 6ml nutrient broth respectively.

Second step added quantitative testing drug respectively in each Erlenmeyer flask, its content is 6MIC concentration, and the growth control group adds the deionized water of equal volume.

In the 3rd step, adding 60 μ l concentration in each Erlenmeyer flask is the to be measured bacterium liquid of 0.5 Maxwell than turbid standard, and each ultimate density of managing bacterium liquid is about 10 ⁶CFU/ml.

The 4th step added behind the bacterium immediately with 4 even vortex 15s of Erlenmeyer flask, after 1000 times of the 10 multiple proportions serial dilutions, getting 0.1ml bacterium liquid respectively is inoculated on the M-H agar plate and (respectively inoculates two parts), evenly be coated with inoculation with L type glass rod, do enumeration after 35 ℃ of overnight incubation, and get its mean number.

The 5th step, cultivate 0.5,1,2,4 respectively for 37 ℃, behind the 6h, with 4 even vortex 15s of Erlenmeyer flask, the same method 10 multiple proportions serial dilutions 10 ⁰～10 ⁷Doubly, get 0.1ml bacterium liquid respectively and be inoculated on the M-H agar plate and (respectively inoculate three parts), after 35 ℃ of overnight incubation, get the plate count of colony number between 30-300, and get its mean number.

In the 6th step, the bacterium colony mean number * extension rate of three blocks of plates of CFU=is in the stoste viable count in every milliliter of bacterium liquid.The bacterium colony of naked eyes counting different time points, and ask its mean value, thus draw the killing curve figure of different time points at logarithmic paper.

The result as can be seen, compares with control group as shown in Figure 8, and the growth of the MRSE after the DS34 effect is subjected to obvious suppression.Along with the prolongation of action time, the colony number of the MRSE of DS34 group reduces gradually; The colony number of ceftazime group MRSE increases gradually, but lacks than the blank group; The growth of Ampicillin Trihydrate group MRSE is compared with the blank group does not have evident difference.Illustrate DS34 to MRSE have fast, tangible killing action, and MRSE has produced serious resistance to ceftazime and Ampicillin Trihydrate.

The mensuration of embodiment 3 antibacterial peptide concentration effects relation.

The first step is got 5 of sterile test tube, is labeled as " growth control pipe, high concentration medicine, middle concentration medicine, lower concentration medicine and Ampicillin Trihydrate group " respectively.

Second goes on foot, and adds the medicine of the different concns of 1ml in each Erlenmeyer flask respectively, and the growth control group adds the deionized water of equal volume.

In the 3rd step, adding 1ml concentration in each Erlenmeyer flask is the to be measured bacterium liquid of 0.5 Maxwell than turbid standard, and each ultimate density of managing bacterium liquid is about 5 * 10 ⁷CFU/ml.

The 4th step is behind 37 ℃ of cultivation 1h, with 5 even vortex 15s of test tube, 10 multiple proportions serial dilutions 10 ⁰～10 ⁷Doubly, get 0.1ml bacterium liquid respectively and be inoculated on the M-H agar plate and (respectively inoculate three parts), after 35 ℃ of overnight incubation, get the plate count of colony number between 30-300, and get its mean number.

In the 5th step, the bacterium colony mean number * extension rate of three blocks of plates of CFU=is in the stoste viable count in every milliliter of bacterium liquid.The bacterium colony of naked eyes counting different concns group, and ask its mean value, thus on logarithmic paper, draw the effect curve of different pharmaceutical concentration.

The result as shown in Figure 9, as can be seen, the CFU of three dosage groups of DS34 (4,12 and 24 μ g/ml) MRSE has reduced by one to two order of magnitude, decrement is all up to (P＜0.01) more than 95%; Along with the increase of DS34 concentration, the CFU of MRSE reduces obvious more.And the CFU of Ampicillin Trihydrate group (Ampicillin Trihydrate) MRSE compares the minimizing (P＞0.05) of no significance with the blank group, does not have significant difference.Experimental result shows that DS34 has tangible killing action to MRSE, and has dose-dependence; And the Ampicillin Trihydrate does not have killing action to MRSE.

The mensuration of the hemolytic toxicity of embodiment 4 antibacterial peptides.

The first step, get the Freshman peripheral blood of anti-freezing, the centrifugal 10min of 3000r/min, draw the hemocyte of lower floor after the layering, add that 0.01M PBS damping fluid is resuspended, washing to be to remove blood plasma and pale brown look blood rete, repeat 3 times, be resuspended at last among the 0.01M PBS of different volumes, obtain 4% and 20% red cell suspension.

Second goes on foot, and gets the antibacterial peptide (the red corpuscle final concentration is 2% and 10%) of the resuspended red corpuscle of 100 μ l and two times of gradient dilutions of 100 μ l, hatches in 37 ℃ incubator.

In the 3rd step, behind the 1h that reaction solution is centrifugal, the 540nm place surveys its optical density value.

The 4th step, the OD that red cell suspension and equal-volume 0.01M PBS are hatched ₅₄₀As 0% haemolysis, the OD of hatching with equal-volume Triton-100 (final concentration 1%) ₅₄₀100% contrast.With antibacterial peptide concentration is X-coordinate, and Δ OD% is the ordinate zou curve plotting, tries to achieve 50% hemolytic antibacterial peptide concentration HC ₅₀

The result as shown in figure 10, as can be seen, antibacterial peptide DS34 and Melittin and HRBC suspension (final concentration is 2% and 10%)

hatch

1,3 altogether, behind the 6h, haemolysis does not all take place in DS34 when 128 μ g/ml, and tangible hemolytic reaction just takes place in Melittin (HRBC suspension final concentration is 2%) when 2.5 μ g/ml.The HC of DS34 ₅₀Far above positive control antibacterial peptide Melittin.

50% plasma stability of embodiment 5 antibacterial peptides is measured.

The first step is got the Freshman peripheral blood of anti-freezing, and the centrifugal 10min of 3000r/min draws upper plasma after the layering, freeze in-20 ℃.Diluting with 0.01M PBS before the test is 50% blood plasma.

In second step, antibacterials and 0.5ml 50%

blood plasma hatch

0,3 respectively at 37 ℃, 6h, and the concentration of antibacterials is 64MIC.

The 3rd step, the MIC of the medicine that detection and blood plasma are hatched altogether.Compare with bacterial growth characteristic in the growth control hole, the lowest concentration of drug of no naked eyes visible growth is for measuring medicine to detecting the MIC of bacterium.

1) the gradient dilution method of antibacterials: take by weighing the 2mg antibacterials and add mixing in the 1ml diluent, the medicine original liquid concentration is 2mg/ml.Use the filtration method degerming after stoste is diluted, packing is standby in a small amount.Stoste can preserved 3 months below-20 ℃, but can only preserve a week under 4 ℃.512 μ l stostes add in the 488 μ l M-H broth cultures, and the maximum concentration that mixes the back antibacterials is 2048 μ g/ml.The medicine of drawing 100 μ l maximum concentrations adds every row in No. 1 hole, No. 1 aperture mixes back sucking-off 100 μ l, add in No. 2, successively behind the doubling dilution to No. 10, sucking-off 100 μ l discard, and the gradient concentration of medicine is followed successively by 1024,512,256,128,64,32,16,8,4,2 μ g/ml like this.

2) detect the preparation of bacterium and standard bacterium: get frozenly in-20 ℃ detection bacterium, streak inoculation is hatched 20～24h in the M-H nutrient agar in incubator (35 ± 2 ℃).Next day, picking mono-clonal bacterium colony was inoculated in the 2ml nutrient broth, and 180rpm, 37 ℃ shake bacterium and spend the night.Be in the bacterium bacterium liquid of logarithmic phase, than turbid standard, use M-H meat soup dilution in 1: 100 (bacteria containing amount about 10 again with M-H broth culture corrected concentrations to 0.5 Maxwell ⁶CFU/ml).Bacterium liquid after the dilution should be inoculated in 15min.The set-up procedure of standard bacterium escherichia coli ATCC25922 and streptococcus aureus ATCC29213 is the same.

3) inoculation of detection bacterium and standard bacterium: the bacterium liquid of getting after 100 μ l dilute with micro sample adding appliance is added in 96 orifice plates successively, and final inoculated bacteria concentration is every hole 5 * 10 ⁵/ ml.At this moment be followed successively by 512,256,128,64,32,16,8,4,2,1 μ g/ml from the 1st hole to the 10 hole drug concentrations.96 orifice plates are put the 1min that vibrates on the microoscillator, make bacterium liquid mixing in each hole, microwell plate is added a cover with minimizing and is hatched evaporation in the process, and puts in the wet box 35 ℃ and hatch 16～20h.Establish no medicine growth control hole (promptly only add the bacterium liquid after 100 μ l M-H meat soups and 100 μ l recover in the 11st hole, do not add medicine) and aseptic control wells (promptly only adding 200 μ l M-H meat soups in the 12nd hole) for every group simultaneously.

4) result judges: aseptic control wells should remain limpid in entire test, shows that whole experiment is aseptic technique.Compare and judge with bacterial growth characteristic in the growth control hole (be muddy shape as meat soup in the micropore, occur precipitation etc. at the bottom of the hole), the lowest concentration of drug of no naked eyes visible growth is for measuring medicine to detecting the MIC of bacterium.

The result as shown in figure 11, as can be seen, after DS34 and 50% blood plasma were hatched, the MIC value was constant, showed that DS34 is stable in blood plasma; S4 (1-16) is hatched back MIC with blood plasma and is obviously increased, and illustrates that it is degraded in blood plasma.

The resistance of embodiment 6 antibacterial peptides is measured.

The first step is measured the MIC value of antibacterials to MRSE, and method is the same.

Second step, the bacterium above the dilution in the 1/2MIC hole, making its concentration is 5 * 10 ⁵CFU/ml, this is the inferior foster bacterium of being commissioned to train.Measure the MIC value of antibacterials to MRSE once more, method is the same.

The 3rd step, continuous 15 days, measure antibacterials MIC value every day to MRSE, method is as above.

In the 4th step, calculate antibacterials to 15 the be commissioned to train relative values of MIC of foster bacterium of the MIC and the 1st that support bacterium of being commissioned to train.

The result as shown in figure 12, as can be seen, MRSE and antibacterials Erythromycin (erythromycin), Ceftriaxone (ceftriaxone), Oxazacillin (Oxacillin) and DS34 are hatched altogether, and continuous 15 the be commissioned to train relative values of foster MIC of foster MIC and the 1st of being commissioned to train are respectively 100,10,10 and 1.Show that MRSE produces resistance to microbiotic easily, be not easy DS34 is produced resistance.

Claims

One group of novel cyclized antibiotic that contains the gamma-core block (DS01～DS37), it is characterized in that, its aminoacid sequence is:

Formula Chinese and English abbreviation expression: Arg arginine, Asn l-asparagine, Cys halfcystine, Gln glutamine, Gly glycine, the Ile Isoleucine, Lys Methionin, Met methionine(Met), Phe phenylalanine, Pro proline(Pro), the Ser Serine, Thr Threonine, Tyr tyrosine, Val Xie Ansuan
2. polypeptide according to claim 1 is characterized in that: DS02～DS07 is the replacement design to DS01, promptly with 6 natural amino acid that contain 4 amino acid replacements of gamma-core block antibacterial peptide intermediary DS1 peptide sequence corresponding site.
3. polypeptide according to claim 2 is characterized in that: DS08～DS13 is respectively to the displacement of DS02～DS07 design, is template with DS02～DS07 peptide sequence promptly, makes the halfcystine of sequence both sides move one to the centre respectively.
4. polypeptide according to claim 1, it is characterized in that: DS14～DS19 is to the brachymemma of DS01 and replaces design, promptly clip preceding 8 amino acid of DS01N-end, make its length foreshorten to 13 amino acid, and naturally contain the amino acid that 4 amino acid of gamma-core block antibacterial peptide intermediary are replaced DS1 peptide sequence corresponding site with 6 by 21 amino acid.
5. polypeptide according to claim 4 is characterized in that: DS20～DS25 is respectively to the displacement of DS14～DS19 design, is template with DS14～DS19 peptide sequence promptly, makes the halfcystine of sequence both sides move one to the centre respectively.
6. polypeptide according to claim 1, it is characterized in that: DS26～DS31 is to the brachymemma of DS01 and replaces design, promptly clip preceding 8 amino acid of DS01N-end and the methionine(Met) of C-end, make its length foreshorten to 12 amino acid, and naturally contain the amino acid that 4 amino acid of gamma-core block antibacterial peptide intermediary are replaced DS1 peptide sequence corresponding site with 6 by 21 amino acid.
7. polypeptide according to claim 6 is characterized in that: DS32～DS37 is respectively to the displacement of DS26～DS31 design, is template with DS26～DS31 peptide sequence promptly, makes the halfcystine of sequence both sides move one to the centre respectively.
8. according to claim 2,4,6 described antibacterial peptides, should comprise with all gamma-core blocks that contains in the gamma-core block antibacterial peptide and form the function equivalent polypeptide that amino acid is replaced, replaced and obtain the sequence that other natural antibacterial peptide carries out; According to claim 3,5,7 described antibacterial peptides, should comprise other amino acid brachymemma and displacement mode and the function equivalent polypeptide that obtains.
9. according to any one polypeptide in the claim 1, wherein related polypeptide be with the solid state chemistry synthetic method produce, the air oxidation process cyclisation, its C-terminal all carries out amidation.
10. according to the antibacterial peptide of claims 1 described anti-multi-drug resistant bacteria, it is characterized in that: described antibacterial peptide sequence is used to prepare the purposes of anti-multi-drug resistant bacteria medicine.